Master of Science (M.Sc.) Theses and Dissertations

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    Open Access
    Molecular characterization of antibiotic resistance in escherichin coil and klebsiella pneumoniae from selected livestock farms in Osun State, Nigeria.
    (Department of Microbiology, Faculty of Science, Obafemi Awolowo University, 2022) SULAYMAN Taofiq Adewale
    This study isolated Escherichia coli and Klebsiella pneumoniae from rectal and faecal samples of some selected livestock farms in Osun State, Nigeria, investigated the antibiotic susceptibility characteristics and the multiple antibiotic resistance index (MARI) phenotype properties of the confirmed isolates, as well as determined the occurrence of some clinically important resistant genes in the isolates. These were with a view to providing information for the mapping of antimicrobial resistance in the agroecosystem. A total of 197 samples were collected from apparently healthy cattle (13 faecal), pigs (61 rectal), and poultry (123 faecal) from 12 selected livestock farms within Osun State. Rectal swabs were collected by gently inserting moistened sterile swab sticks into the rectum of the animals while fresh faecal droppings were scooped with sterile swab sticks. E. coli and K. pneumoniae were presumptively isolated based on cultural characteristics. The identity of the presumptive isolates was confirmed using polymerase chain reaction detection of the uidA (E. coli) and ITS (K. pneumoniae). Antibiotic susceptibility of the isolates was determined using the disk diffusion method based on the interpretative standard described by the Clinical Laboratory Standard Institute. The presence of genes encoding clinically significant resistance determinants was also determined using polymerase chain reaction for the isolates. A total of 238 presumptive E. coli isolates and 123 presumptive Klebsiella pneumoniae isolates were recovered from the collected samples. Out of the presumptive isolates, 207 (87%) and 66 (53.7%) were confirmed as E. coli and K. pneumoniae, respectively, using polymerase chain reaction. However, 32 (48.5%) non-repetitive K. pneumoniae isolates and 90 (43.5%) non-repetitive E. coli isolates were selected for further analysis. For the E. coli isolates, a high resistance rate was observed against tetracycline (81.1%), ciprofloxacin (40%) and ampicillin (36.7%). In the K. pneumoniae isolates, high resistance rates were observed against ampicillin (78.1%), ciprofloxacin (65.6%) and tetracycline (50%). The multiple antibiotic resistancepattern of the isolates revealed resistance against at least three different antibiotics, while the multiple antibiotic resistance indices ranged from 0.3 to 0.8. Genes encoding beta-lactamases: blaTEM (83.3%), blaOXA-1-LIKE (37.8%), blaSHV (4.4%) blaFOX (68.9%), blaACC (45.6%), blaCIT (41.1%), blaMOX (10%), blaDHA (2.2%), blaEBC (2.2%), and plasmid mediated quinolone resistance genes: qepA (25.6%), aac(6’)-Ib-cr (16.7%), oqxB (64.4%), oqxA (16.7%), qnrB (2.2%), qnrA (14.4%) and qnrS (45.6%) were detected in the E. coli isolates, while, blaTEM (78.1%), blaOXA-1-LIKE (75%), blaSHV (68.8%) blaFOX (75%), blaACC (34.4%), blaCIT (6.3%), qepA (18.8%), aac(6’)-Ib-cr (21.9%), oqxB (100%), oqxA (71.9%), qnrB (9.4%), and qnrS (28.1%) were detected in the K. pneumoniae isolates. This study concluded that multidrug-resistant E. coli and K. pneumoniae, harbouring clinically significant resistance genes are present in livestock which could be transferred to humans through direct contact or the food chain. Plasmid-mediated quinolone resistance and beta-lactamases genes, often reported in clinical settings, were detected in some of the isolates in this study.
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    Open Access
    Production, purification and characterization of keratinase from keratinase producing bacteria isolated from selected chicken Feature dumpsites in Ile Ife Nigeria
    (Department of Microbiology, Faculty of science, Obafemi Awolowo Universty, Ile Ife, 2021) Oluwatobi Sanson Ayegun
    This research study focused on the isolation and identification of keratinase-producing bacteria from soil samples of selected chicken feather dump sites. It also explored the optimum conditions required for the production of keratinase by the isolated keratinase producing bacterium, thereby leading to the partial purification and characterization of the keratinase produced using its physicochemical and kinetic parameters. Soil samples were obtained from selected feather dumpsites in Ile-Ife from where keratinase producing bacteria isolated and purified using nutrient agar. The isolates were screened on skimmed milk agar and the isolates with high yield were subjected to submerged fermentation in order to select the best bacterial isolate for keratinase production. After 72 hours of incubation at a temperature of 37 oC, the mineral salt media were centrifuged at 4000 rpm for 20 minutes in a cold condition and the supernatants obtained were used as crude enzymes which were later quantified using keratinase assay and protein concentration. The optimum conditions of various parameters were investigated for the optimal production of keratinase by the isolate. Afterwards, all the optimum parameters were combined and used for the bulk production of crude enzyme. The crude enzyme produced was partially purified using 90 % Ammonium sulphate saturation and ion exchange chromatography and the partially purified enzyme was later characterized. A total of 110 bacteria were isolated and screened for keratinase production, 98 of the bacterial isolates tested positive to the screening process using Skimmed Milk agar. The best keratinase producer using the results of the keratinase assay and protein concentration from the submerged fermentation process was molecularly identified as Bacillus subtilis 2C-9B. The optimal temperature required for the production of keratinase by Bacillus subtilis 2C-9B was discovered to be 35 oC and at a pH 9.0. The carbon and nitrogen source that gave an optimal production of keratinase was discovered to be feather meal and yeast extract respectively. The bulk enzyme produced was partially purified using 90 % ammonium sulphate saturation and the result gave a 0.68-fold purification and this was further purified by ion exchange chromatography yielding an active protein peak at 4.9-fold purification. The purification process was followed by the characterization of the partially purified keratinase. An optimum pH in the alkaline region of pH 11.0 and optimum temperature of 45ºC was recorded. Metal ions like Ba2+, Hg2+ and Mn2+ inhibited the enzyme activity whereas Cu2+ and Pb2+ exhibited a slight inhibition. Phenylmethylsulphonyl fluoride (PMSF) completely inhibited the activity of the partially purified keratinase. However, EDTA did not inhibit the enzyme which implies that the partially purified enzyme belongs to the serine protease family and not the metalloprotease family. The study concluded that the keratinase produced by the isolated Bacillus subtilis 2C-9B strain was an alkalophilic enzyme which implies that it is suitable for industrial applications in leather processing and detergent industry.
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    Open Access
    Production and purification of xylanase from a fungus isolated from a paper industry solid waste dumpsite in Osogbo,Osun State, Nigeria
    (Department of Microbiology, Faculty of Science, Obafemi Awolowo Universty, 2023) Salawudeen, Morufat Tolani
    This study isolated and screened for xylanolytic fungi from a paper industry solid waste dumpsite. It also identified the selected fungus and then optimized the process parameters for xylanase production from selected fungus and to produced, purified as well as characterized xylanase produced from fungi isolated from a paper industry solid waste dumpsite. These were with a view to providing useful information on the potential utilization of agricultural waste as a substrate for the production of xylanase to fulfill various industrial needs. Soil samples were collected aseptically from four different location at a paper industry waste dumpsite in Osogbo, Osun State. The samples collected was processed for isolation using spread plate method and standard methods were used for characterizing and identifying the fungal isolates, which were subsequently subjected to screening using both rapid plate assay method and submerged fermentation for the process of producing xylanase. The fungus with the best xylanase activity was selected for enzyme production, purification and characterization. The best optimal conditions for xylanase synthesis by the best producing fungus were determined by varying the nitrogen sources, pH, carbon sources, temperature, inoculum volumes and different concentrations of the carbon and nitrogen sources for media composition. The best optimal condition were used for bulk synthesis of xylanase to produce the crude enzyme. The crude enzyme was partially purified using a one step purifications method of ion exchange chromatography using CM-Sephadex C-50. The effect of pH, temperature, metal ions, heat stability were studied and the kinetic parameters (Km and Vmax) of the partially purify xylanase were determined. The selected fungus, SD5, has a zones of hydrolysis and highest enzyme activity of about 12 mm and 212.30 Units/ml/min respectively from the 32 isolates obtained from the samples collected from the paper waste dumpsites after been screened. The fungus SD5 was molecularly characterized and identified as Aspergillus oryzae strain KhA0707 with 98% identity. With an inoculum amount of 1.0 ml and 4 days of fungal growth, the enzyme production reached its peak. The best optimal temperature and pH of production was 30°C and pH 6.0 respectively, while the best nitrogen and carbon sources are peptone and xylan respectively. The partially purified enzyme showed that specific activity was 5437.35 units/mg with purification fold of 12.33. Characterization of the partially purified xylanase showed that the optimum activity occurred at temperature and pH of 55 °C and 6.0 respectively. The enzyme kinetic Vmax value is 161.290 units/ml/min and Km value of 0.6129 mg/ml. EDTA totally inhibited the activity of the partially purified xylanase. The enzyme activity was slightly inhibited by Ca2+, Na+, even at 1 mM while K+, Cu2+, Al3+, urea and Mecarptopyruvate promote the xylanase activity. The findings of this research showed that a paper waste dumpsite is also a potential source for xylanolytic fungus. The isolated Aspergillus oryzae employed for xylanase production in this study was able to generate a significant quantity of xylanase, which will be useful in protein hydrolysis and industries.
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    Open Access
    Isolation, characterization and bioactivity studies of the extract from aerial part of hilleria latifolia (LAM)
    (Department of Chemistry, Faculty of Science, Obafemi Awolowo University, Ile-Ife., 2023) Arowosegbe, Sunday Micheal
    This study screened the crude extract of Hilleria latifolia for its phytochemical constituents and evaluated the extract and fractions for their antioxidant and antibacterial activities. It also isolated compound from its active fraction and elucidated the structure of the compound. The antioxidant and antibacterial potential of the isolated compound were also determined. These were with a view to finding novel bioactive compound from this medicinal plant. The aerial parts of the plant was air-dried and pulverized. The pulverized plant was extracted with 90% methanol/water for 96 hours and filtered. The filtrate obtained was concentrated to dryness in vacuo at 40 ℃. This afforded the crude extract which was subjected to solvent partitioning using solvents of varying polarities: n-hexane, dichloromethane and ethyl acetate in turn. This yielded four solvent fractions, and the aqueous MeOH fraction inclusive. The crude and the solvent fractions were tested for antibacterial activity, Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and antioxidant activities using a number of antioxidant assays namely: 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Nitric Oxide (NO) Scavenging, Metal Chelating Ability (MCA), Ferric Reducing Antioxidant Power (FRAP) and Total Antioxidant Capacity (TAC). The antibacterial activity was evaluated using the Agar diffusion method. The results showed that of all the solvent fractions obtained, the ethyl acetate fraction was the most active in the DPPH, TAC, MCA, and FRAP assays. One compound was isolated from the aqueous fraction using column chromatography. The isolated compound, HLA-1 was characterized by Nuclear Magnetic Resonance (NMR) and Infrared (IR) spectroscopy. Compound HLA-1 was identified as 8 – methyl – 1, 4 – oxazo – 2 – enine – 5, 7, 9 – trione. The n–hexane and dichloromethane fraction obtained from the crude extracts of Hilleria latifolia exhibited significant biological and antioxidant activities. These fractions were subjected to GC – MS analyses and a total of 27 compounds were identified in both fractions. Most of the identified constituents have been reported to possess various biological activities such as antioxidant, anticancer, anti-inflammatory and antibacterial activities. The study concluded that the extracts and isolated compound from Hilleria latifolia exhibited varying degree of antibacterial and antioxidant activities and could be effective in the management of infectious and oxidative stress related diseases. These observed activities could justify the ethnomedicinal uses of the plant.
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    Open Access
    Comparative assessment of plant-based extracts and synthetic insecticides on the growth, yield and proximate composition of cowpea (Vigna unguiculata L. Walp)
    (Institute of Ecology and Environmental Studies, Faculty of Science, Obafemi Awolowo University., 2022) FOYINSOLAMI Ebunoluwa Adebayo
    This study assessed the effect of plant-based and synthetic insecticides application on the growth response and yield of cowpea. It also investigated the effects of the insecticides application on the biomass, grain yield and proximate composition of cowpea grains produced. These were with a view to providing information on the use of plant-based extracts on quantity and quality of cowpea grains. The experiment was conducted on a vacant land measuring 11.75 m x 7.50 m behind the Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife. Viable seeds of Ife-brown cultivar of cowpea were purchased from the Institute of Agricultural Research and Training, Ibadan. The experimental location was cleared manually two times using a cutlass and hand-held hoe. The experiment consisted of four treatments which were: extracts of three plant-based (Azadirachta indica, Tithonia diversifolia, Chromolaena odorata) and cypermethrin that served as control. The extracts of fresh shoots of A. indica, T. diversifolia, and C. odorata were separately prepared using standard method. The experiment was made up of 12 plots, each measuring 1.5 m x 2.0 m and the plots were arranged in a randomized complete block design. Four seeds per stand were sown using 50 cm x 30 cm spacing and seedlings thinned to 2 stands per hole at 2 weeks after sowing (WAS). The plots were weeded at 3 and 6 WAS. The cowpea stands were sprayed with plant-based and cypermethrin using the rates 100 g/L/plot and 15 mL/L/plot respectively at 5, 6, 7 and 8 WAS. Growth parameters such as plant height, number of leaves and stem girth were measured bi-weekly from 2 to 8 WAS and extent of leaf damage at 7 and 8 WAS. Tagged cowpea stands were carefully uprooted at 10 WAS to determine the total biomass yield. Cowpea pods were harvested when the pods turned yellow at 10 WAS and threshed. Proximate composition of cowpea grains (crude protein, ash, fibre, carbohydrate, fat and dry matter), and post-cropped soil analyses were carried out using standard methods. Data obtained were subjected to analysis of variance and their treatment means were separated using Tukey’s multiple comparison test at p < 0.05. The results showed that pH of the pre-cropped soil was 6.94 and soil texture was loamy sand. Organic carbon and total nitrogen were 0.64 and 5.82 g/kg, respectively and these values reduced to between 13 and 30% across the treatment plots for the post-cropped soil. The growth parameters; height (cm), number of leaves and stem girth (cm) at 6 WAS were: 23.06 ± 0.86, 55.29 ± 4.59 and 1.58 ± 0.05 for A. indica; 24.15 ± 0.75, 57.58 ± 3.94 and 1.69 ± 0.06 for T. diversifolia; 21.76 ± 0.68, 48.38 ± 2.15 and 1.55 ± 0.05 for C. odorata; and 21.99 ± 1.18, 45.26 ± 3.45 and 1.46 ± 0.07 for cypermethrin, respectively. Also, the grain yield of cowpea with cypermethrin, 1.08 t/ha was significantly (p < 0.05) higher than the best plant-based insecticide, T. diversifolia. Cowpea grains obtained with T. diversifolia and C. odorata had comparable and high values of crude protein (29.7%, 28.3%) and fibre (6.1%, 6.5%) respectively. The study concluded that T. diversifolia compared favourably with cypermethrin, in terms of grain yield of cowpea, whereas T. diversifolia and C. odorata gave higher proximate composition of cowpea.